Linköping University Medical Dissertations No.530

Insulin control of glucose transport in caveolae
microdomains of the plasma membrane

Johanna Gustavsson

Akademisk avhandling
som för avläggande av medicine doktorsexamen vid Linköpings Universitet kommer att offentligt försvaras i Berzeliussalen, Hälsouniversitetet, fredagen den 10 oktober 1997, kl. 09.00.
Fakultetesopponent: Docent Harriet Wallberg-Henriksson, Avd för fysiologi, Karolinska Sjukhuset, Stockholm

Abstract

Caveolae are invaginated, dynamic micro-domains in the plasma membrane and believed to be involved in receptor-mediated uptake of small molecules (potocytosis) and in signal transduction. A phosphatidylinositol glycan, a precursor of potential insulin second messengers, has been found to be enriched in the caveolae-fraction of adipocyte plasma membranes (Parpal et al., 1995, J Cell Biol 131:125-135). We now demonstrate that the insulin receptor is localized to caveolae micro-domains. This was investigated in i) 3T3-L1 adipocyte plasma membranes by a morphological method (double immunofluorescence labeling and confocal microscopy) and in ii) caveolae isolated by a biochemical, detergent-free method. The insulin receptor was enriched in caveolae, and, in response to insulin, the receptor tyrosine kinase was activated since the insulin receptor in caveolae was tyrosine-phosphorylated .

Insulin stimulates the translocation of glucose transporter proteins from intracellular stores to the plasma membrane, leading to an increased glucose uptake. Long-chain 1,2-diacylglycerol, one of two potential second messengers for insulin, has been found to stimulate glucose uptake in rat adipocytes (Strålfors, 1988, Nature, 335:554-556). Here, we report that long-chain 1,2-diacylglycerol, emulsified in taurodeoxycholate, stimulates the translocation of GLUT4 to the plasma membrane. Physiological long-chain diacylglycerols are taken up in amounts sufficient to have biological effects, equally well in the absence of taurodeoxycholate.

We also report that the rapid translocation of GLUT4 to the plasma membrane was followed by a slower transition of GLUT4 into caveolae. The accumulation of GLUT4 in caveolae coincided with the insulin-stimulated increase in glucose uptake. This offers a mechanistic explanation for the observed discrepancies between the appearance of GLUT4 in the plasma membrane and the delayed increase in glucose uptake.

Non-hydrolyzable GTP-analogues stimulate the translocation of GLUT4 and increase glucose uptake in permeabilized cells. The small GTP-binding protein Rab4 is suggested to be involved in these processes since Rab4 has been localized to GLUT4-containing vesicles and is redistributed in response to insulin. We found that Rab4 is enriched in caveolae and that the amount of Rab4 increased in caveolae, in the same order of magnitude as GLUT4 did, in response to insulin.

Caveolae are characterized by high levels of sphingolipids and cholesterol. Depletion of cholesterol, which disrupts the integrity of caveolae, abolished insulin-stimulated glucose uptake reversibly. Insulin's control of protein phosphorylation was also abolished while beta-adreneric signaling was unaffected.

The results suggest that caveolae are crucial for insulin-regulated events in adipocytes and implicate that disruption of these structures could have consequences for the development of insulin resistance and diabetes mellitus.

Department of Biomedicine and Surgery, Division of Cell Biology
Faculty of Health Sciences, S-581 85 Linköping, Sweden
Linköping 1997

ISBN 91-7871-794-9